BLOOD UREA, Estimation of Blood Urea, by Berthelot Reaction, by Diacetyl Monoxime Method Reagents

 BLOOD  UREA, Estimation of Blood Urea, by Berthelot Reaction, by Diacetyl Monoxime Method  Reagents

 

UREA - Urea constitutes almost half the total of the nonprotein nitrogenous substances of the blood. It is manufactured in the liver from carbon dioxide and ammonia resulting from the breakdown of amino acids. It is the major excretory product of protein metabolism. 

Urea is carried by the plasma to the kidney where it is filtered from the plasma by the glomerulus. About 40 % of the urea in the glomerular filtrate is reabsorbed by the renal tubules. Most of the urea in the filtrate is excreted in urine while small amounts are excreted through the gastrointestinal tract and the skin.

Measurements of urea were initially done on a protein free filtrate of whole blood and so, early analytical methods were based on measuring the amount of nitrogen. 

This analysis was commonly referred to as Blood Urea Nitrogen (BUN). Though current methods of urea estimation use whole blood, plasma or serum, the term BUN is still used by some workers.

The nitrogen concentration of urea can be converted to urea concentration by multiplying it by 2.14.

The concentration of urea nitrogen in mg/dl can be converted to mmol/l of urea by multiplying by 0.36 

Collection of specimens Since urea estimation can be performed on whole blood, plasma or serum, any routine anticoagulant such as EDTA can be used except

(i) Sodium fluoride, because it is an enzyme inhibitor, and therefore, unsuitable for enzyme methods.

(ii) Ammonium oxalate, because some methods of estimation of urea depend on the measurement of ammonia. 

Choice of methods There are three commonly used methods for the estimation of blood urea. 

1. Urease method using Nessler's reaction The enzyme urease hydrolyses urea to form ammonium carbonate. The aminonium ions produced are measured by using Nessler's reagent.

 

2. Urease method using Berthelot reaction In this method, the ammonia produced by the action of urease on urea is estimated by measuring the blue colour of indophenol formed with phenol and hypochlorite.Sodium nitroprusside is used as a catalyst. 

3. Diacetyl monoxime method Urea is heated with diacetyl monoxime in the presence of a strong acid and an oxidizing agent to form a yellow diazine derivative. Thiosemicarbazide is added to the mixture to enhance and stabilise the colour. Methods (2) and (3) will be described in detail.

Estimation of Plasma Urea by Berthelot Reaction

Reagents

(i) Buffered urease reagent: 1.0g ethylenediaminetetraacetic acid (EDTA) and 150 mg urease are mixed and shaken in about 70 ml of distilled water. Adjust pH to 6.5 and make up volume to 100 ml. When stored in a plastic bottle at 4°C, the reagent is stable for one month. 

(ii) Phenol colour reagent Dissolve 50 g analytical grade phenol in 400 ml distilled water in a volumetric flask. Dissolve separately 250 mg analytical grade sodium nitroprusside in 50 ml of distilled water. Mix the two solutions together and make the volume up to 1 litre with distilled water. Store in a dark brown bottle away from light at 4°C. Stability is for about 2 months.

(iii) Alkaline hypochlorite reagent Dissolve 25 g sodium hydroxide in 400 ml distilled water. Dissolve 2.1 g sodium hypochlorite in 100 ml of distilled water, mix the two solutions and make up the volume to 1 litre. Storage is same as for the phenol colour reagent.

(iv) Urea standard solution (10 mmol/l) Dissolve 600 mg urea in distilled water and make up to 1 litre. Add a few drops of chloroform as a 

preservative. Store at 4°C.

Method

(a) Set up test tubes as follows:

	Blank	Test	Standard
Buffered urease reagent	200 ul	200 ul	200 ul
Test plasma		20 ul
Standard			20 ul
Distilled water	20 ul

(b) Incubate all tubes at 37°C for 15 min.

(c) Remove all tubes from the water bath; add 1.0 ml phenol color reagent to each tube.Shake gently to mix.

(d) Add 1.0 ml hypochlorite reagent to each tube and mix.

(e) Incubate all tubes at 37°C for 20 minutes.

(f) Remove all the tubes from water bath; add 5 ml distilled water to each tube.

(g) Read absorbance of the standard and test Against the blank at 630 nm to 650 nm or with red filter

Estimation of Blood Urea by Diacetyl Monoxime Method

Reagents

 (I)Sodium tungstate (10 %)

(II) 2/3 N sulphuric acid Add 2 ml of conc. H2SO4 to about 50 ml distilled water and dilute up to 100 ml.

(III) Color reagent

Solution A Dissolve I g diacetyl monoxime, 0.2 g thiosemicarbazide and 9 g sodium chloride in distilled water. Dilute up to 1 litre.

Solution B  Add 60 ml conc. H_SO, and 10 ml of 85% phosphoric acid to 800 ml of distilled water. Dissolve 0.1 g ferric chloride in the acid mixture; cool and make up to 1 litre. 

Reagent mixture Mix solution A and solution B in equal volumes just before use.

(vi) Urea standard solution (5 mmol/L) Dissolve 300 mg urea in distilled water and make up to 1 litre.

Method 

(i) Prepare protein-free filtrate by mixing 1.0ml of blood, 7.0 ml of distilled water. Mix and add 1.0 ml of sodium tungstate and 1.0 ml of 2/3 N H 30 Mix and allow to stand for 5 minutes. Filter or centrifuge.

(ii) Set up 3 tubes as follows:

	blank	Standard 5.0 ml	Test 5.0 ml
Color reagent	5.0 ml	5.0 ml	5.0ml
Distilled water	0.5 ml
Urea standard -		0.5 ml
Specimen(filtrate)			0.5 ml

(iii) Heat all the tubes in boiling water bath for exactly 15 minutes.

(iv)  Cool and read the absorbance at 520 nm or using green filter.

Alternatively, a calibration curve can be prepared and a graph of absorbance against concentration plotted to determine the amount of urea in the test sample.

Normal Range of Blood Urea

Adults: 2.5 - 7.0 mmol/L (15 - 40 mg/dl)

Mg/dl of urea = mmol/L x 6

Note Urea concentration is often expressed as units of urea nitrogen in blood (BUN) in mg/dl. The molecular weight of urea is 60. Each molecule of urea contains two nitrogen atoms with a weight of 14 x 2 = 28. Therefore blood urea can be converted to BUN by the factor 28/60, i.e., 0.467. Conversely, BUN can be converted to urea by multiplying by 60/ 28, i.e., 2.14.

Interpretation of Blood Urea Results

Blood normally contains a small amount of urea; and the amount may vary with the amount of protein in the diet. When the amount of urea in the blood becomes excessive, the condition is known as uraemia. This condition is usually a result of impaired kidney function. In the elderly, the level of urea may be a little higher than normal; and low values may be found during pregnancy and in full term infants, whereas premature infants may have slightly higher values than the adult range.

A considerable rise in the blood NPN level particularly due to urea and creatinine in a kidney insufficiency is called azotaemia. The cause of azotaemia can be pre-renal, renal, or post-renal. 

Pre-renal azotaemia This is due to poor perfusion of the kidneys resulting in diminished glomerular filtration. Poor perfusion can be due to:

(a) Reduced blood flow to the kidney as a result of shock, dehydration, blood loss or congestive heart failure. 

(b) Increased protein catabolism as in fever,severe burns, stress, crush injury or bleeding into soft tissues.

Renal azotaemia This is when urea is retained primarily due to impaired glomerular filtration which results in acute or chronic renal disease. The acute state may be due to glomerulonephritis, nephrotoxic drugs, or renal cortical necrosis. The chronic condition may be due to glomerulonephritis, pyelonephritis, diabetes mellitus or renal tubular diseases. 

Post-renal azotaemia This is due to the obstruction of where urea is reabsorbed into circulation. The obstruction can be due to ureteral destruction by stone, tumour, inflammation or surgical trauma. It can also be due to obstruction of bladder neck or urethra by enlarged prostate gland, tumour. inflammation or stones.

Note

Semi-quantitative methods for the estimation of urea are available. One of such methods is the Urastrat which is based on a test strip coated with the urease reagent. The methods are useful in emergencies, small clinics or as screening procedures.

Apart from the measurement of the amount of urea in the blood, there are many methods for determining the impairment of the kidneys. But because most of the methods are cumbersome, they are not used routinely.

Determination of Urine Urea

Collect a 24 hour specimen of urine, which is preserved with thymol or toluene. Dilute the urine 1:20 and determine the urea concentration of the diluted urine using any method described for blood urea. Multiply the results by the dilution factor (x 20) to obtain the concentration in mmol/L or g/dl. 

Normal range 250-600 mmol/L or 15-35 g/dl 

Urea clearance test The average urea clearance over a 24 hour period can be determined by collecting a 24 hour urine specimen and taking a blood sample around mid-day. Determine the plasma and urine urea concentrations and calculate average urea clearance in the same manner as the creatinine clearance (see below).

Normal range 40-65 ml/min. It may fall to about 10ml/min during renal failure and may reach to 2-3 ml/min in terminal cases.

Note

Urea clearance test is no longer used as a measure of glomerular filtration rate (GFR) because it underestimates GFR.